聶洪港，劉冉冉，楊悠悠，劉虎威，白　玉

(1.北京大學(xué)分析測(cè)試中心，北京　100871；2.北京分子科學(xué)國(guó)家實(shí)驗(yàn)室，生物有機(jī)與分子工程教育部重點(diǎn)實(shí)驗(yàn)室，北京大學(xué)化學(xué)與分子工程學(xué)院分析化學(xué)研究所，北京　100871；3.中糧營(yíng)養(yǎng)健康研究院，營(yíng)養(yǎng)健康與食品安全北京市重點(diǎn)實(shí)驗(yàn)室，北京　102209)

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二維液相色譜-質(zhì)譜法研究犬腎小管上皮細(xì)胞脂質(zhì)組成及馬兜鈴酸(Ⅰ)對(duì)其影響

聶洪港1，劉冉冉2，楊悠悠3，劉虎威2，白玉2

(1.北京大學(xué)分析測(cè)試中心，北京100871；2.北京分子科學(xué)國(guó)家實(shí)驗(yàn)室，生物有機(jī)與分子工程教育部重點(diǎn)實(shí)驗(yàn)室，北京大學(xué)化學(xué)與分子工程學(xué)院分析化學(xué)研究所，北京100871；3.中糧營(yíng)養(yǎng)健康研究院，營(yíng)養(yǎng)健康與食品安全北京市重點(diǎn)實(shí)驗(yàn)室，北京102209)

摘要：采用在線正反相二維液相色譜-質(zhì)譜聯(lián)用技術(shù)研究了犬腎小管上皮細(xì)胞脂質(zhì)組成及馬兜鈴酸(Ⅰ)對(duì)其影響。二維色譜的第一維用于分離不同種類(lèi)脂質(zhì)，第二維用于分離同類(lèi)脂質(zhì)的不同分子，進(jìn)而利用高分辨質(zhì)譜對(duì)脂質(zhì)分子進(jìn)行檢測(cè)。該方法減少了共流出，降低了電離抑制，提高了靈敏度與準(zhǔn)確性。借助精確質(zhì)荷比檢索數(shù)據(jù)庫(kù)、高分辨二級(jí)質(zhì)譜和當(dāng)量碳數(shù)與保留時(shí)間規(guī)律等方法檢測(cè)了犬腎小管上皮細(xì)胞中13類(lèi)脂質(zhì)的1 416個(gè)脂質(zhì)分子。選取11種外源性脂質(zhì)標(biāo)準(zhǔn)品進(jìn)行方法驗(yàn)證，方法的線性關(guān)系、檢測(cè)限、重復(fù)性均滿足檢測(cè)要求。在此基礎(chǔ)上，考察了犬腎小管上皮細(xì)胞暴露于馬兜鈴酸(Ⅰ)后的脂質(zhì)變化情況，對(duì)改變含量2～4倍的15個(gè)脂質(zhì)分子進(jìn)行了鑒定。該實(shí)驗(yàn)結(jié)果可為馬兜鈴酸的毒理、病理研究和相關(guān)疾病的臨床診斷提供豐富的信息，并展現(xiàn)了二維液相色譜-質(zhì)譜法在脂質(zhì)組學(xué)研究中廣闊的應(yīng)用前景。

關(guān)鍵詞：二維液相色譜-質(zhì)譜；犬腎小管上皮細(xì)胞；馬兜鈴酸；脂質(zhì)組學(xué)

網(wǎng)絡(luò)出版時(shí)間：2016-07-05；網(wǎng)絡(luò)出版地址：http:∥www.cnki.net/kcms/detail/11.2979.TH.20160705.1012.002.html

馬兜鈴酸(aristolochic acid，AA)是從馬兜鈴屬植物中提取出的一系列硝基苯蒽環(huán)類(lèi)羧酸，主要成分是馬兜鈴酸(Ⅰ)和馬兜鈴酸(Ⅱ)，其結(jié)構(gòu)示于圖1。古人用馬兜鈴屬植物治療蛇咬傷，現(xiàn)代醫(yī)學(xué)則利用AA的抗炎性能治療關(guān)節(jié)炎、痛風(fēng)、風(fēng)濕以及膿瘡等[1-2]。但有研究證明，AA是造成馬兜鈴酸腎病的原因，該病是長(zhǎng)期攝入馬兜鈴屬植物后發(fā)生的一種快速進(jìn)行性纖維化間質(zhì)腎炎[3-4]，其病理表現(xiàn)有腎臟體積縮小、部分兩側(cè)腎臟不對(duì)稱(chēng)、外形不規(guī)整，常伴有腎小管萎縮和腎小管消失等[5]。該病變以皮質(zhì)淺層受累最為明顯，越往皮質(zhì)深層病變?cè)捷p。探求馬兜鈴酸腎病的藥理、毒理和作用機(jī)制一直是該領(lǐng)域的研究熱點(diǎn)，代謝組學(xué)在相關(guān)研究中發(fā)揮了重要作用[6-7]。

圖1　馬兜鈴酸(Ⅰ)和馬兜鈴酸(Ⅱ)的結(jié)構(gòu)式[8]

脂質(zhì)組學(xué)是代謝組學(xué)最為活躍的分支之一，它通過(guò)系統(tǒng)全面地分析生物體中脂質(zhì)分子的種屬、代謝情況，以及與其他分子的相互作用，揭示其在生命活動(dòng)中對(duì)膜的構(gòu)建、信號(hào)傳導(dǎo)、調(diào)控轉(zhuǎn)錄和翻譯等過(guò)程的影響，闡明脂質(zhì)及其代謝與細(xì)胞、器官和機(jī)體的生理、病理之間的關(guān)系[9-11]。色譜、質(zhì)譜及相關(guān)聯(lián)用技術(shù)是脂質(zhì)組學(xué)研究最主要的分析技術(shù)[12-13]。其中，在線正反相二維液相色譜-質(zhì)譜聯(lián)用技術(shù)可實(shí)現(xiàn)在一次進(jìn)樣中先分離不同種類(lèi)脂質(zhì)，再分離鑒定同類(lèi)脂質(zhì)中的不同分子，可最大限度地減少共流出、降低電離抑制、增加低豐度脂質(zhì)分子信息、減少假陽(yáng)性的檢測(cè)結(jié)果、提升脂質(zhì)分子鑒定的準(zhǔn)確性和信息量，已被應(yīng)用于多種樣品的脂質(zhì)組學(xué)研究[14-19]。

本工作擬采用二維液相色譜-質(zhì)譜聯(lián)用方法檢測(cè)腎小管上皮(Madin-Darby canine kidney, MDCK)細(xì)胞中磷脂、脂肪酸、鞘脂等脂質(zhì)成分，并將該細(xì)胞暴露于AA(Ⅰ)中，考察上述脂質(zhì)化合物含量的變化情況，以期從脂質(zhì)組學(xué)角度為AA(Ⅰ)的腎毒理學(xué)研究和馬兜鈴酸腎病的臨床診斷提供依據(jù)。

1實(shí)驗(yàn)部分

1.1主要儀器與裝置

自建二維液相色譜系統(tǒng)：第一維采用美國(guó)Agilent公司的1100液相色譜儀，配有四元泵、在線脫氣機(jī)、自動(dòng)進(jìn)樣器、柱溫箱；第二維采用美國(guó)Agilent公司的1200液相色譜儀，配有二元泵和在線脫氣機(jī)；兩維色譜之間的接口包括美國(guó)IDEX Health & Science公司的兩位六通閥，英國(guó)Edwards 公司的E2M2真空泵和自制的電熱水浴鍋。Agilent6530四極桿飛行時(shí)間質(zhì)譜儀：美國(guó)Agilent公司產(chǎn)品，配有噴射流離子聚焦技術(shù)電噴霧離子源。

儀器控制及數(shù)據(jù)采集由美國(guó)Agilent公司MassHunter Data Acquisition B.02.00完成；定性、定量及統(tǒng)計(jì)學(xué)分析分別由美國(guó)Agilent公司MassHunter Qualitative Analysis B.02.00、MassHunter Quantitative Analysis B.03.01和Mass Profiler Professional 2.0完成。

1.2主要材料與試劑

馬兜鈴酸(Ⅰ)標(biāo)準(zhǔn)品：由中國(guó)藥品生物制品檢定所提供；磷脂標(biāo)準(zhǔn)品：sn-(1-heptade-canoyl-2-hydroxy)-glycerol-3-phospho-sn-3′-(1′,2′-heptadecanoyl)-glycerol，Hemi BMP(17∶0)；1-(10Z-heptadecenoyl)-sn-glycero-3-phospho-(1′-rac-glycerol) (sodium salt)，LPG(17∶1)；1,2-dimyristoyl-sn-glycero-3-phospho-(1′-rac-glycerol) (sodium salt)，PG(14∶0/14∶0)；N-(dodecanoyl)-heptadecasphing-4-enine-1-phosphoethanolamine，Sphingosyl PE(d17∶1/12∶0)；1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine，PE(14∶0/14∶0)； 1-(10Z-heptadecenoyl)-sn-glycero-3-phosphoethanolamine，LPE(17∶0)；1,2-dimyristoyl-sn-glycero-3-phospho-L-serine (sodium salt)，PS(14∶0/14∶0)；N-heptadecanoyl-D-erythro-sphingosylphosphorylcholine，SM (d18∶1/17∶0)；1-heptadecanoyl-sn-glycero-3-phosphocholine，LPC(17∶0)；1,2-dimyristoleoyl-sn-glycero-3-phosphocholine，PC (14∶0/14∶0)：美國(guó)Avanti Polar Lipid公司產(chǎn)品；十七烷酸(heptadecanoic acid)標(biāo)準(zhǔn)品FFA(17∶0)、質(zhì)譜純甲酸銨、細(xì)胞培養(yǎng)級(jí)二甲基亞砜：均為美國(guó)Sigma-Aldrich公司產(chǎn)品；正己烷、乙醇、甲醇和氯仿：均為色譜純，美國(guó)Dikma公司產(chǎn)品；實(shí)驗(yàn)用純凈水：中國(guó)娃哈哈公司產(chǎn)品；MDCK細(xì)胞：美國(guó)American Type Culture Collection (ATCC)公司產(chǎn)品；DMEM高糖培養(yǎng)基、10%胎牛血清、1%谷胺酰胺、1%青霉素/鏈霉素和胰酶/EDTA消化液：均為美國(guó)Thermo-Fisher公司產(chǎn)品。

1.3樣品制備

1.3.1MDCK細(xì)胞培養(yǎng)MDCK細(xì)胞采用DMEM高糖培養(yǎng)基培養(yǎng)，內(nèi)含10%胎牛血清、1%谷胺酰胺、1%青霉素/鏈霉素，于5%CO2、恒濕和37 ℃條件下培養(yǎng)，待90%融合后，用胰酶/EDTA消化液消化，制成單細(xì)胞懸液；調(diào)節(jié)細(xì)胞密度為每毫升5×105個(gè)，以每孔1 mL接種于6孔細(xì)胞培養(yǎng)板，24 h后棄去培養(yǎng)液，用PBS清洗2次；向給藥組的每個(gè)孔內(nèi)分別加入200 nmol AA(Ⅰ)，與未給藥對(duì)照組一同置于5%CO2、37 ℃培養(yǎng)箱中，繼續(xù)培養(yǎng)24 h；24 h后，給藥組的部分細(xì)胞死亡，懸浮在培養(yǎng)基里，部分仍貼壁；收集每個(gè)樣品的培養(yǎng)液，并用胰蛋白酶消化仍然貼壁的細(xì)胞，將收集的貼壁細(xì)胞分別用血球計(jì)數(shù)板計(jì)數(shù)。

1.3.2MDCK細(xì)胞脂質(zhì)的提取采用經(jīng)改進(jìn)的Folch方法[20]提取MDCK細(xì)胞脂質(zhì)。首先從給藥組和對(duì)照組各選取8份細(xì)胞樣品，每份樣品含有約5×105個(gè)活細(xì)胞，向其中加入10 mL Folch溶劑(氯仿-甲醇溶液，2∶1，V/V)，以3 000 r/min離心10 min，取下層溶液，氮?dú)獯蹈?，?fù)溶于1 mL環(huán)己烷-異丙醇溶液(7∶3,V/V)中，待色譜-質(zhì)譜分析。

1.3.3方法驗(yàn)證樣品的制備 隨機(jī)選取10份細(xì)胞樣品(每份含約5×105個(gè)活細(xì)胞)，向1號(hào)樣品中加入11種脂質(zhì)標(biāo)準(zhǔn)品各0.001 μg，而2～10號(hào)樣品中11種脂質(zhì)標(biāo)準(zhǔn)品的加入量分別為0.002、0.01、0.02、0.1、0.2、1、2、10、20 μg。采用1.3.2節(jié)方法，得到10個(gè)方法驗(yàn)證用樣品，其單一內(nèi)標(biāo)濃度分別為0.001、0.002、0.01、0.02、0.1、0.2、1、2、10、20 mg/L。

1.4實(shí)驗(yàn)條件

1.4.1色譜條件第一維色譜柱為Agilent Rx-SIL色譜柱(2.1 mm×150 mm×5 μm)，柱溫25 ℃，進(jìn)樣量20 μL；流動(dòng)相：A1為含有5 mmol/L甲酸銨的環(huán)己烷-異丙醇-水溶液(30∶70∶2，V/V/V)，B1為含有5 mmol/L甲酸銨的甲醇-水溶液(100∶2，V/V)，流速0.1 mL/min。

第二維色譜柱為Agilent Eclipse Plus C8色譜柱(2.1 mm×10 mm×3.5 μm)，柱溫40 ℃；流動(dòng)相：A2為含有5 mmol/L甲酸銨的甲醇-水溶液(50∶50，V/V)，B2為含有5 mmol/L甲酸銨的甲醇，流速0.3 mL/min。

接口水浴鍋溫度50 ℃，在真空泵的作用下，一維色譜流出的流動(dòng)相在接口處氣化并被真空泵抽出，被分析物質(zhì)沉積在樣品環(huán)中，當(dāng)六通閥切換時(shí)，其被轉(zhuǎn)移至第二維色譜系統(tǒng)進(jìn)行進(jìn)一步的分離。第一維和第二維液相色譜梯度洗脫條件和六通閥的轉(zhuǎn)換時(shí)間列于表1。

1.4.2質(zhì)譜條件負(fù)離子模式；鞘氣溫度350 ℃，鞘氣流速8 L/min；噴霧氣壓力138 kPa；干燥氣溫度300 ℃，干燥氣流速5 L/min；毛細(xì)管入口電壓3 500 V；碎裂電壓190 V；錐孔1電壓65 V；質(zhì)譜采集速率1.02 spectra/s，采集范圍為m/z100～2 000；在目標(biāo)二級(jí)質(zhì)譜模式中，母離子質(zhì)量窗口m/z1.3，碰撞能量40 V。

2結(jié)果與討論

2.1MDCK細(xì)胞脂質(zhì)的分離與檢測(cè)

MDCK細(xì)胞中的脂質(zhì)提取物在第一維色譜中按其極性差異實(shí)現(xiàn)分離，共檢測(cè)到13類(lèi)脂質(zhì)：游離脂肪酸(FFA)，磷脂酰甘油(PG)，降解磷脂酰甘油(LPG)，磷脂酰肌醇(PI)，降解磷脂酰肌醇(LPI)，磷脂酰乙醇胺(PE), 降解磷脂酰乙醇胺(LPE)，磷脂酰絲氨酸(PS)，降解磷脂酰絲氨酸(LPS)，磷脂酰膽堿(PC)，降解磷脂酰膽堿(LPC)，鞘磷脂(SM)和心磷脂(CL)。將第一維色譜分為5段(第1段含F(xiàn)FA；第2段含PI、PG、LPG；第3段含PE、CL；第4段含LPI、LPE、PS、LPS；第5段含PC、LPC、SM)，分別轉(zhuǎn)移至第二維反相色譜進(jìn)行進(jìn)一步的分離，結(jié)果示于圖2。

表1　二維液相色譜梯度洗脫程序

圖2　MDCK細(xì)胞脂質(zhì)的一維(a)和二維(b)液相色譜基峰色譜圖

將下面3種方式相結(jié)合對(duì)脂質(zhì)化合物進(jìn)行定性：1) 根據(jù)精確質(zhì)量數(shù)檢索脂質(zhì)數(shù)據(jù)庫(kù)，推測(cè)分子式；2) 根據(jù)脂質(zhì)二級(jí)質(zhì)譜特征碎片離子解析脂質(zhì)分子結(jié)構(gòu)。根據(jù)文獻(xiàn)報(bào)道[21-29]及本實(shí)驗(yàn)室前期工作經(jīng)驗(yàn)[14-19]，除FFA外，其余12類(lèi)脂質(zhì)化合物在豐度滿足二級(jí)質(zhì)譜分析的前提下，均可得到具有特征性的二級(jí)離子，適用于脂類(lèi)化合物的結(jié)構(gòu)解析，主要脂質(zhì)化合物的離子碎片列于表2；3) 根據(jù)同類(lèi)脂質(zhì)分子中支鏈的當(dāng)量碳數(shù)與其保留時(shí)間的關(guān)系。

對(duì)于大多數(shù)含量相對(duì)較高的非脂肪酸類(lèi)脂質(zhì)化合物，可以采用精確質(zhì)荷比、化合物特征碎裂途徑和保留時(shí)間共同定性；而低豐度的脂質(zhì)分子和脂肪酸類(lèi)化合物通常因?yàn)闊o(wú)法得到穩(wěn)定可靠的二級(jí)質(zhì)譜信息，則需通過(guò)精確質(zhì)荷比和保留時(shí)間進(jìn)行定性，下面將分別介紹。

首先，將一級(jí)質(zhì)譜檢測(cè)得到的準(zhǔn)分子離子質(zhì)荷比輸入LIPIDMAPS網(wǎng)站(http:∥www.lipidmaps.org/tools/ms/LMSD_search_mass_options.php)的在線數(shù)據(jù)庫(kù)“Search the LMSD for lipids with a given mass (m/z) value”進(jìn)行搜索，數(shù)據(jù)庫(kù)軟件會(huì)列出可能的脂類(lèi)化合物。Q-TOF MS在有參比離子校正的情況下，質(zhì)量誤差可控制在±0.002以下；而在沒(méi)有參比離子進(jìn)行校正的二級(jí)質(zhì)譜中，質(zhì)量誤差可達(dá)±0.005以下。與理論計(jì)算值相比，相對(duì)質(zhì)量誤差在±0.005以上則認(rèn)為未能達(dá)到定性要求，因此本實(shí)驗(yàn)設(shè)定的質(zhì)量窗口是±0.005。例如，在第3段檢測(cè)到某一分析物離子的質(zhì)荷比為766.540 6，將這一數(shù)值輸入在線數(shù)據(jù)庫(kù)進(jìn)行檢索。因?yàn)樵诘?段流出的脂質(zhì)分子屬于PE類(lèi)化合物，而在數(shù)據(jù)庫(kù)檢索結(jié)果中既符合PE類(lèi)脂質(zhì)，又符合質(zhì)量偏差小于±0.005的只有C43H77NO8P-PE(38∶4) (理論質(zhì)量數(shù)為766.539 2)，所以基本可以確定該脂質(zhì)是兩脂肪酸支鏈上含有38個(gè)碳原子和4個(gè)不飽和雙鍵的PE分子丟失一個(gè)H+形成的準(zhǔn)分子離子，同時(shí)可確定這一化合物的分子式。若要確定脂肪酸鏈的具體信息，還需要對(duì)該化合物的二級(jí)質(zhì)譜進(jìn)行解析。在結(jié)構(gòu)分析的過(guò)程中，采用targeted MS/MS模式選取母離子(m/z766.54)，以40 V的碎裂能量對(duì)母離子進(jìn)行碎裂，采集碎片離子信息，得到其二級(jí)質(zhì)譜圖，示于圖3。圖中，m/z303.232 9和m/z283.263 4離子分別是20∶4和18∶0脂肪酸根的離子；m/z480.309 7 和m/z462.298 7離子分別對(duì)應(yīng)于PE(38∶4)負(fù)離子以烯酮和脂肪酸的形式中性丟失一個(gè)20∶4脂肪?；鶄?cè)鏈后形成的離子；m/z196.036 6和m/z140.011 2離子是所有PE分子的特征碎片離子；m/z196.036 6代表PE準(zhǔn)分子離子的兩個(gè)脂肪酰基側(cè)鏈，其中一個(gè)以脂肪酸的形式，另一個(gè)以烯酮的形式全部中性丟失后產(chǎn)生的特征離子；m/z140.011 2離子則是乙醇胺磷酸負(fù)離子。這些碎片離子的信息與文獻(xiàn)[21-29]報(bào)道一致，因此可推斷出這一分子的兩個(gè)脂肪酰基的組成是18∶0和20∶4，脂質(zhì)分子可確定為PE(18∶0/20∶4)。大部分脂質(zhì)分子的碎裂機(jī)理已被詳細(xì)研究過(guò)，根據(jù)這些脂質(zhì)分子的特征碎裂路徑和產(chǎn)生的特征離子，可以對(duì)產(chǎn)生穩(wěn)定可靠二級(jí)質(zhì)譜的高豐度化合物進(jìn)行定性分析。

表2　主要脂質(zhì)化合物的離子碎片

由于脂肪酸類(lèi)化合物和其他豐度相對(duì)較低的脂質(zhì)化合物很難得到穩(wěn)定的二級(jí)質(zhì)譜信息，因此，本實(shí)驗(yàn)采用在數(shù)據(jù)庫(kù)中搜索檢測(cè)到的準(zhǔn)分子離子質(zhì)荷比以確定其可能的分子信息，并結(jié)合保留時(shí)間進(jìn)行輔助定性。實(shí)驗(yàn)發(fā)現(xiàn)，同類(lèi)脂質(zhì)分子在反相色譜分離中的保留時(shí)間與其支鏈的當(dāng)量碳數(shù)相關(guān)，支鏈上含有相同碳原子個(gè)數(shù)的同類(lèi)脂質(zhì)分子隨著支鏈上碳碳雙鍵個(gè)數(shù)的增加疏水性減弱，在反相色譜柱上的保留時(shí)間縮短，這與文獻(xiàn)[14]發(fā)現(xiàn)的規(guī)律一致。利用這個(gè)規(guī)律，可以對(duì)無(wú)法得到二級(jí)質(zhì)譜信息的脂類(lèi)化合物進(jìn)行輔助定性，這比僅使用精確的準(zhǔn)分子離子質(zhì)量對(duì)化合物定性更加準(zhǔn)確。

通過(guò)上述3種方式對(duì)MDCK細(xì)胞脂質(zhì)提取物的二維液相色譜-質(zhì)譜結(jié)果進(jìn)行分析，在13類(lèi)脂質(zhì)中共檢測(cè)到1 416個(gè)脂質(zhì)分子，結(jié)果列于表3。其中，PC和PE是檢出脂質(zhì)分子個(gè)數(shù)最多的兩類(lèi)脂質(zhì)化合物，占總個(gè)數(shù)的68%；其他11類(lèi)脂質(zhì)分子僅占總個(gè)數(shù)的32%。

圖3　負(fù)離子模式下，母離子m/z 766.54的二級(jí)質(zhì)譜圖

脂質(zhì)種類(lèi)PIPGPCPEPSLPILPGLPCLPELPSCLSMFFA總數(shù)脂質(zhì)分子個(gè)數(shù)8810490473771375644195530541416

2.2方法驗(yàn)證

從11類(lèi)脂質(zhì)中各選取1個(gè)外源性脂質(zhì)標(biāo)準(zhǔn)品，使其保留時(shí)間較均勻地分布于第一維色譜中，5段中每段含有2～3個(gè)脂質(zhì)標(biāo)準(zhǔn)品：第1段含F(xiàn)FA(17∶0)和Hemi BMP(17∶0)；第2段含LPG(17∶1)和PG(14∶0/14∶0)；第3段含PE(14∶0/14∶0)和Sphingosyl PE(d17∶1/12∶0)；第4段含LPE(17∶0)和PS(14∶0/14∶0)；第5段含 SM (d18∶1/17∶0)、LPC(17∶0)和PC (14∶0/14∶0)。采用這11個(gè)脂質(zhì)標(biāo)準(zhǔn)品，按2.4節(jié)方法配制10個(gè)濃度水平的驗(yàn)證樣品，每個(gè)濃度水平連續(xù)檢測(cè)3次，且每個(gè)濃度水平之間插入一次空白溶劑的分析。計(jì)算時(shí)對(duì)測(cè)試標(biāo)準(zhǔn)品的萃取離子色譜峰進(jìn)行積分，得到該化合物相應(yīng)濃度的峰面積，將每個(gè)濃度水平測(cè)得的3次峰面積取平均值與相應(yīng)的濃度建立線性方程。計(jì)算得到的11個(gè)脂質(zhì)標(biāo)準(zhǔn)品的線性方程、線性范圍、線性回歸系數(shù)R2、檢測(cè)限(LOD)、相對(duì)標(biāo)準(zhǔn)偏差(RSD)等，列于表4?？芍?1種驗(yàn)證標(biāo)準(zhǔn)品的線性回歸系數(shù)均大于0.991 7，表明11種標(biāo)準(zhǔn)品在相應(yīng)的范圍內(nèi)質(zhì)譜信號(hào)響應(yīng)和濃度具有良好的線性關(guān)系。除PS(14∶0/14∶0) 的LOD為5 μg/L外，其余10種脂質(zhì)標(biāo)準(zhǔn)的LOD都達(dá)到2 μg/L，峰面積和保留時(shí)間的相對(duì)標(biāo)準(zhǔn)偏差分別小于7.7%和0.11%(n=6)，這說(shuō)明本方法可滿足定量比較分析的需要，適用于脂質(zhì)組學(xué)的相關(guān)研究。

表4　11種標(biāo)準(zhǔn)品的方法驗(yàn)證結(jié)果

注：1.y為萃取離子峰的峰面積，x為標(biāo)準(zhǔn)品濃度(μg/L)；2.標(biāo)準(zhǔn)品濃度0.1 mg/L，n=6；3.萃取離子色譜圖的峰面積

2.3馬兜鈴酸(Ⅰ)對(duì)MDCK細(xì)胞中脂質(zhì)含量的影響

采用Mass Profiler Professional軟件對(duì)給藥組和對(duì)照組各8份細(xì)胞脂質(zhì)提取物的二維液相色譜-質(zhì)譜結(jié)果進(jìn)行統(tǒng)計(jì)學(xué)分析，主成分分析(PCA)結(jié)果示于圖4?？梢?jiàn)，16個(gè)樣品被分成2組，該軟件給出了區(qū)分這2組數(shù)據(jù)做出主要貢獻(xiàn)的化合物列表，通過(guò)對(duì)這些化合物設(shè)定閾值，即峰面積的絕對(duì)倍數(shù)變化大于2且p值小于0.05，共找到2組中峰面積存在顯著差異的15個(gè)脂質(zhì)分子，結(jié)果列于表5。

采用四極桿飛行時(shí)間質(zhì)譜對(duì)上述15個(gè)離子進(jìn)行二級(jí)碎裂，由碎片離子推測(cè)可能的碎裂路徑，進(jìn)一步確認(rèn)其可能的分子組成與結(jié)構(gòu)信息。以LPE(O-16∶1)為例，其二級(jí)質(zhì)譜圖和碎裂路徑示于圖5。

圖4　用AA(Ⅰ)處理的MDCK細(xì)胞和對(duì)照組數(shù)據(jù)的主成分分析圖

脂質(zhì)分子保留時(shí)間/minm/z變化規(guī)律變化倍數(shù)FFA(20∶4)22.870303.2320up2FFA(24∶1)26.214365.3418up4LPG(22∶6)39.736555.2732up3LPG(20∶4)39.738531.2733up2～3LPG(16∶0)39.908483.2725up2～4LPG(18∶1)40.168509.2885up2LPG(O-18∶1)40.653495.3080up2LPI(20∶3)88.948621.3035up2LPE(20∶5)88.978498.2616up2～3LPE(20∶4)89.353500.2776up2LPE(22∶4)89.988528.3086up2LPE(O-16∶1)90.044436.2832up2LPE(O-18∶1)91.072464.3144up2PC(O-36∶6)128.987762.5438up2～3PC(O-36∶5)131.596764.5589up2～3

圖5　LPE(O-16∶1)的二級(jí)質(zhì)譜圖和碎裂路徑

15個(gè)脂質(zhì)分子的含量在給藥組中有明顯變化，這可能同AA(Ⅰ)與MDCK細(xì)胞的DNA形成加合物，其轉(zhuǎn)錄成的蛋白對(duì)脂質(zhì)合成的調(diào)控能力降低有關(guān)；也可能是AA直接與相關(guān)蛋白作用，改變其活性，致使脂質(zhì)合成不受控，脂質(zhì)的大量合成占用了過(guò)多的能量，造成了細(xì)胞凋亡，或超量的脂質(zhì)影響了細(xì)胞的正常代謝使其凋亡。上述推斷仍需進(jìn)一步研究的驗(yàn)證。給藥前后含量發(fā)生明顯變化的脂質(zhì)分子可能會(huì)由細(xì)胞滲入尿液，使其在尿液中的含量增加，這有望為臨床診斷馬兜鈴酸腎病提供一種無(wú)損檢測(cè)的方法。

4總結(jié)與展望

本工作采用二維液相色譜-質(zhì)譜法分析了MDCK細(xì)胞中13類(lèi)脂質(zhì)共1 416種分子種屬，并比較了這些脂質(zhì)在MDCK暴露于AA(Ⅰ)前后含量的變化，為毒理學(xué)研究和潛在臨床診斷方法提供了技術(shù)手段。二維液相色譜-質(zhì)譜法在生物脂質(zhì)輪廓分析，尤其是低豐度脂質(zhì)分析中，具有靈敏度高、定性準(zhǔn)確、信息豐富等特點(diǎn)，有望在脂質(zhì)組學(xué)研究中發(fā)揮更加重要的作用。

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中圖分類(lèi)號(hào)：O657.63

文獻(xiàn)標(biāo)志碼：A

文章編號(hào)：1004-2997(2016)04-0289-12

doi：10.7538/zpxb.youxian.2016.0037

收稿日期：2016-01-11;修回日期：2016-03-05

基金項(xiàng)目：國(guó)家自然科學(xué)基金項(xiàng)目(21175005，21405006，21527809)資助

作者簡(jiǎn)介：聶洪港(1980—)，男(漢族)，遼寧丹東人，博士，從事色譜、質(zhì)譜儀器及檢測(cè)技術(shù)研究。E-mail: hgnie@pku.edu.cn

通信作者：白玉(1976—)，女(漢族)，吉林九臺(tái)人，副教授，從事生物分離與檢測(cè)研究。E-mail: yu.bai@pku.edu.cn

Lipid Profiling of Madin-Darby Canine Kidney Cells and Its Lipid Changes Induced by Treatment of Aristolochic Acid (Ⅰ) Using Two Dimensional Liquid Chromatography-Mass Spectrometry

NIE Hong-gang1, LIU Ran-ran2, YANG You-you3, LIU Hu-wei2, BAI Yu2

(1.AnalyticalInstrumentationCenter,PekingUniversity,Beijing100871,China;2.BeijingNationalLaboratoryforMolecularSciences,KeyLaboratoryofBioorganicChemistryandMolecularEngineeringofMinistryofEducation,InstituteofAnalyticalChemistry,CollegeofChemistryandMolecularEngineering,PekingUniversity,Beijing100871,China;3.BeijingKeyLaboratoryofNutritionHealthandFoodSafety,ChineseOil&FoodstuffsCorporation(COFCO)NutritionandHealthResearchInstitute,Beijing102209,China)

Abstract:The mechanisms of lipids separation are as following: different lipid classes are separated by adsorption mechanisms and eluted out of the column in normal-phase liquid chromatography (NPLC), and individual molecular species are separated based on hydrophobicity in reversed-phase liquid chromatography (RPLC). In RPLC, the elution sequence of lipid molecules is determined by both the chain length and the degree of unsaturation in the fatty-acyl chains. To avoid co-elution of molecular species, an online, normal-phase and reversed-phase two-dimensional (2D) liquid chromatography (LC) quadrupole time-of-flight mass spectrometry (Q TOF-MS) system was developed for the lipid profiling of Madin-Darby canine kidney (MDCK) cells and the investigation of the lipid changes in MDCK cells treated with aristolochic acid (Ⅰ). Different lipid classes in MDCK cells were separated in the first dimension of the two-dimensional liquid chromatograph system and lipid molecular species were further separated in the second dimension followed by mass spectrometry detection, so that the ion suppression effects were reduced while the detection sensitivity was improved.

All lipids in MDCK cells were identified with high accuracy mass values measured by Agilent 6530 accurate mass Q TOF-MS. The abundant molecular species were confirmed by targeted MS/MS，meanwhile the retention time and low abundance lipid molecules were identified withm/zvalue and the retention time based on the correlation between the equivalent carbon number (ECN) and the retention time. The measured accurate masses were applied for preliminary identification using the online database with a mass tolerance of less than ±0.005 on the basis of the predicted elemental composition. 1 416 endogenous lipid species from 13 lipid classes were identified by accurate masses, tandem mass spectra and the retention time. 11 exogenous lipid standards from different classes, including FA 17∶0, Hemi BMP(17∶0), LPG(17∶1 ), PG(14∶0/14∶0), Sphingosyl PE(d17∶1/12∶0), PE(14∶0/14∶0), LPE(17∶1), PS(14∶0/14∶0), LPC(17∶0), PC(14∶1/14∶1), SM(d18∶1/17∶0), were selected to be separated in five fractions for the evaluation of this method. The linear regression coefficients (R2=0.991 7-0.998 3), the limit of detection (2-5 μg/L) and the relative standard deviation of peak area (0.9%-7.7%) and retention time (0.01%-0.11%) were all satisfactory.

To investigate the lipid changes in MDCK cells dosed with aristolochic acid (Ⅰ), 16 MDCK cell samples (each containing 5×105cells) were randomly separated into a dosed group (n=8) and a control group (n=8). All 16 samples were detected by the 2D LC/MS method. The dosed group and control group were alternately injected to reduce systemic error. During the sequence, one blank sample was injected after every three injections, and no significant carryover of lipids was observed. MS data of all 16 samples were extracted by Mass-Hunter Qualitative Analysis software and analyzed by Mass Profiler Professional software. Through setting threshold parameters, the software presented a list of potential biomarkers whose absolute fold-change of peak area was larger than 2 andpvalue less than 0.05. By the above-mentioned approach, 15 changed lipid species were confirmed, as their concentrations in the dosed group were 2-4 times of those in the control group. The results would contribute to the study on therapeutic and toxicological mechanisms of aristolochic acids and revealed that this two-dimensional liquid chromatography quadrupole time-of-flight mass spectrometry method was a promising tool for lipidomics research.

Key words：two-dimensional liquid chromatography mass spectrometry; Madin-Darby canine kidney (MDCK) cells; aristolochic acid; lipidomics